黄瓜雌花花冠蛋白质双向电泳技术体系的建立.PDFVIP

中国农学通报 2016,32(19):51-58 Chinese Agricultural Science Bulletin 黄瓜雌花花冠蛋白质双向电泳技术体系的建立 路 曼，宋晓飞，李晓丽，孙成振，闫立英 （河北科技师范学院园艺科技学院，河北昌黎066600） 摘 要：为建立适合黄瓜(Cucumis sativus L.)雌花花冠蛋白质双向电泳技术体系，以开花当天的黄瓜雌花 花冠为试验材料，从蛋白质提取方法、IPG 胶条梯度、等电聚焦程序、蛋白质上样量、染色方法等方面进 行了探索。结果表明：酚抽提法得到的黄瓜雌花花冠蛋白质纯度高、产量高。17 cm pH 4~7 IPG 胶条得 到的双向电泳图谱蛋白质点分布均匀、清晰。优化后的等电聚焦程序适当增加了低压除盐步骤和时间， 得到的双向电泳图谱蛋白质点圆且多。300 µg 上样量的双向电泳图谱蛋白质点可达400 多个，低丰度 蛋白质点清晰。硝酸银染色的双向电泳图谱蛋白质点较多。该试验获得了蛋白质点分布均匀、背景清 晰、分辨率高、质量高的双向电泳图谱，为研究黄瓜雌花花冠蛋白质组学提供了参考。 关键词：黄瓜；雌花花冠；蛋白质；双向电泳 中图分类号：S642.2 文献标志码：A 论文编号：cas Two-dimensionalElectrophoresisSystemforCucumberFemaleFlower’sCorolla LuMan,SongXiaofei,LiXiaoli,SunChengzhen,YanLiying (CollegeofHorticulturalScience,HebeiNormalUniversityofScienceandTechnology,ChangliHebei066600) Abstract: Anappropriatetwo-dimensionalelectrophoresisproteomeanalysissystemofcucumberfemale flower’scorollawasestablishedbyoptimizingtheparametersincludingtheproteinextractionmethod,thepH gradient of IPG strip, isoelectric focusing conditions, sample application, and dyeing method. The result showed that phenol extraction method could obtain more protein with higher purity. Two-dimensional electrophoresismapsusing 17cmpH4-7IPGstriphadnooverlappingandproteinpointsdistributedevenly. Goodproteinpointswereobtainedinthetwo-dimensionalelectrophoresismapsusingoptimizedisoelectric focusingconditionwithaddedlow-pressuredesalinationstepandtime.Whenthesampleapplicationincreased to 300 μg,thenumberofproteinpointsreached 400 andthelowabundanceproteinpointswerealsoclear. Moreproteinpointswereobtainedinthetwo-dimensionalelectrophoresismapsusingsilverstainingthan colloidCoomassiebrilliantbluestaining.Thisstudyobtainedclearbackground,highresolution,andhigh qualitytwo-dimensionalelectropho